UBCH7-like ubiquitin-conjugating enzyme

ABSTRACT

The present invention provides a human ubiquitin-conjugating enzyme (UBCPB) and polynucleotides which identify and encode UBCPB. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding UBCPB and a method for producing UBCPB. The invention also provides for agonists, antibodies, or antagonists specifically binding UBCPB, and their use, in the prevention and treatment of diseases associated with expression of UBCPB. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding UBCPB for the treatment of diseases associated with the expression of UBCPB. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding UBCPB.

This application is a divisional application of U.S. application Ser.No. 08/748,703, filed Nov. 13, 1996.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of anovel ubiquitin-conjugating enzyme and to the use of these sequences inthe diagnosis, prevention, and treatment of Alzheimer's disease, musclewasting syndrome, viral infections, and cancer.

BACKGROUND OF THE INVENTION

The ubiquitin system is a major pathway for selective proteindegradation (Finley D. et al. (1991) Annu. Rev. Cell Biol. 7: 25-69).Degradation by this system is instrumental in a variety of cellularfunctions such as DNA repair, cell cycle progression, signaltransduction, transcription, and antigen presentation. The ubiquitinpathway also eliminates proteins that are misfolded, misplaced, or thatare in other ways abnormal. This pathway requires the covalentattachment of ubiquitin (E1), a highly conserved 76 amino acid protein,to defined lysine residues of substrate proteins.

Substrate recognition by this pathway involves a specialized recognitionand targeting apparatus, the ubiquitin-conjugating system.Ubiquitin-conjugating enzyme (E2) and ubiquitin-protein ligase (E3),either independently or in conjunction, catalyze isopeptide formationbetween the carboxyl terminus of ubiquitin and amino groups of internallysine residues of target proteins (Scheffner M. et al. (1995) Nature373: 81-83). Ubiquitin-protein conjugates are then recognized anddegraded by a specific protease complex, the 26S proteasome. Both E2 andE3 exist as protein families, and their pattern of expression is thoughtto determine substrate specificity (Nuber U. et al. (1996) J. Biol.Chem. 271: 2795-2800). For example, E6 oncoprotein of thecancer-associated human papillomavirus types 16 and 18, inactivates thetumor suppressor protein p53 via the ubiquitin protein degradationpathway. An E3 protein, E6-AP, and an E2 protein, either UbcH5 or UbcH7,complex with E6 and specifically conjugate ubiquitin to p53 (ScheffnerM. et al. (1993) Cell 75: 495-505; Nuber et al., supra). Other E2proteins are not sufficient for p53 ubiquitination, thus UbcH5 and UbcH7appear to be involved in the specific targeting of p53 for degradation.

The yeast ubiquitin-conjugating enzyme Ubc3 (also known as CDC34) playsa crucial role in the progression of the cell cycle from the G1 to Sphase and the correct positioning of ubiquitin on a surface of Ubc3 is arequirement for this cell cycle transition (Prendergast J. A. et al.(1995) J. Biol. Chem. 270: 9347-9352). Mutation studies have suggestedthat amino acids S-73, S-97, and S-139 of Ubc3 may be critical forsubstrate specificity, while C-95 is the site of catalytic activity (LiuY. et al. (1995) Mol. Cell Biol. 15: 5635-5644). An alteration in C-95and another highly conserved amino acid, L-99, resulted in a dominantnegative mutation (Banerjee A. et al. (1995) J. Biol. Chem. 270:26209-26215). Overexpression of this mutation of Ubc3 was found to blockcell growth in otherwise wild type strains.

A decrease in muscle mass, known as muscle wasting or cachexia, has beenshown to be associated with the ubiquitin-dependent proteolytic system.Rats bearing the Yoshida AH-130 ascites hepatoma for 7 days showed asignificant decrease in muscle mass in relation to non-tumor bearingcontrols (Llovera M. et al. (1995) Int. J. Cancer 61: 138-141). Themuscle wasting was found to be associated with an increased proteolyticrate related to the ubiquitin-dependent proteolytic system. Musclewasting is common among human cancer patients. In addition to cancer,ubiquitin-dependent muscle wasting is also influenced by nutritionalmanipulation (such as fasting and dietary protein deficiency), muscleactivity and disuse, AIDS, and the pathological conditions, sepsis,trauma, and acidosis (Attaix D. et al. (1994) Reprod. Nutr. Dev. 34:583-597). In a rat model for long lasting sepsis, researchers found thatE2 mRNA levels increase during the acute and chronic disease phases andparallel a rise in muscle protein breakdown (Voisin L. et al. (1996) J.Clin. Invest. 97: 1610-1617).

The presence of ubiquitin and ubiquitin conjugates has been detected inpatients affected by neurodegenerative diseases such as Alzheimer'sdisease. Whereas the intracellular amyloid beta-protein precursor (APP)did not show appreciable ubiquitin-mediated degradation, threeextracellular APP forms were degraded by this proteolytic pathway,suggesting a potential regulatory role for the ubiquitin-dependentsystem in the in vivo APP metabolic pathway (Gregori L. et al. (1994)Biochem. Biophys. Res. Commun. 203: 1731-1738). Paired helical filaments(PHF) are fibrillar structures that accumulate in degenerating neuronsin the brains of Alzheimer's disease patients. One component of PHF, thePHF-smear, consists of the tau protein fragment bound to ubiquitin(Morishima M. et al. (1994) Dementia 5: 282-288).

Evidence from experiments on mouse and rabbit reticulocytes indicatethat ubiquitin conjugation is a key rate-limiting step in antigenpresentation (Grant E.P. et al. (1995) J. Immunol. 155: 3750-3758). Therates of degradation of beta-galactosidase constructs correlated withthe rates of class I antigen presentation in vivo. This shows thatubiquitin degradation pathway may have a critical role in generatingmajor histocompatibility complex (MHC) class I-presented peptides.

Depletion of specific cellular proteins may have many medical andagricultural benefits. Redirecting the ubiquitin-dependent proteolyticpathway may facilitate specific proteolytic removal. Gosink M. M. et al.(1995, Proc. Natl. Acad. Sci. 92: 9117-9121) report examples in whichE2s target recognition was redefined by engineering E2s to containappropriate protein-binding peptides fused to their C termini. Thus, itmay be possible to design E2s capable of directing the selective removalof many intracellular proteins, such as those implicated in thepathogenesis of Alzheimer's disease.

The ability to selectively modulate E2 activity may provide the meansfor treating diseases associated with protein degradation, such asAlzheimer's disease, muscle wasting syndrome or a means by whichundesired proteins may be targeted for degradation, such as in the caseof viral infections and cancer. A new E2 having different specificitycould target a unique set of proteins for degradation.

The discovery of polynucleotides encoding ubiquitin-conjugating enzymes,and the enzymes themselves, provides the means to investigate proteindegradation pathways. Discovery of molecules related toubiquitin-conjugating enzyme satisfies a need in the art by providingnew diagnostic or therapeutic compositions useful in monitoring ormodulating protein degradation in patients with Alzheimer's disease,muscle wasting syndrome, viral infections, or cancer.

SUMMARY OF THE INVENTION

The present invention features a novel ubiquitin-conjugating enzymehereinafter designated UBCPB and characterized as having chemical andstructural similarity to UbcH7.

Accordingly, the invention features a substantially purified UBCPB whichhas the amino acid sequence shown in SEQ ID NO:1.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode UBCPB. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

The invention also relates to a polynucleotide sequence comprising thecomplement of SEQ ID NO:2 or variants thereof. In addition, theinvention features polynucleotide sequences which hybridize understringent conditions to SEQ ID NO:2.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode UBCPB. The presentinvention also features antibodies which bind specifically to UBCPB, andpharmaceutical compositions comprising substantially purified UBCPB. Theinvention also features the use of agonists and antagonists of UBCPB.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO: 1) and nucleicacid sequence (SEQ ID NO:2) of UBCPB. The alignment was produced usingMACDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

FIG. 2 shows the amino acid sequence alignments among UBCPB (SEQ IDNO:1), UbcH7 (GI 1064916; SEQ ID NO:3), and UbcH5 (GI 460810; SEQ IDNO:4). The alignment was produced using the multisequence alignmentprogram of DNASTAR™ software (DNASTAR Inc, Madison Wis.).

FIG. 3 shows the hydrophobicity plot (MACDNASIS PRO software) for UBCPB,SEQ ID NO: 1; the positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity.

FIG. 4 shows the hydrophobicity plot for UbcH7, SEQ ID NO:3.

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

Definitions

"Nucleic acid sequence", as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded, and represent the sense or antisense strand. Similarly,"amino acid sequence", as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragments or portionsthereof, and to naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "protein" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

"Peptide nucleic acid", as used herein, refers to a molecule whichcomprises an oligomer to which an amino acid residue, such as lysine,and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63).

UBCPB, as used herein, refers to the amino acid sequences ofsubstantially purified UBCPB obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of UBCPB, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic UBCPB, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to UBCPB, causes a change in UBCPB which modulates the activity ofUBCPB. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to UBCPB.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to UBCPB, blocks or modulates the biologicalor immunological activity of UBCPB. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to UBCPB.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of UBCPB. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of UBCPB.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of UBCPB orportions thereof and, as such, is able to effect some or all of theactions of ubiquitin-conjugating enzyme-like molecules.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding UBCPB or the encoded UBCPB.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification", as used herein, refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen binds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term "homology", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially homologous." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length human UBCPB and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to proteins in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding UBCPB orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2 by northern analysis is indicative of thepresence of mRNA encoding UBCPB in a sample and thereby correlates withexpression of the transcript from the polynucleotide encoding theprotein.

"Alterations" in the polynucleotide of SEQ ID NO: 2, as used herein,comprise any alteration in the sequence of polynucleotides encodingUBCPB including deletions, insertions, and point mutations that may bedetected using hybridization assays. Included within this definition isthe detection of alterations to the genomic DNA sequence which encodesUBCPB (e.g., by alterations in the pattern of restriction fragmentlength polymorphisms capable of hybridizing to SEQ ID NO:2), theinability of a selected fragment of SEQ ID NO: 2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding UBCPB (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab')₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind UBCPBpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

The Invention

The invention is based on the discovery of a novel humanubiquitin-conjugating enzyme, (UBCPB), the polynucleotides encodingUBCPB, and the use of these compositions for the diagnosis, prevention,or treatment of Alzheimer's disease, muscle wasting syndrome, viralinfections, and cancer.

Nucleic acids encoding the human UBCPB of the present invention werefirst identified in Incyte Clone 1614950 from the brain tumor cDNAlibrary (BRAITUT12) through a computer-generated search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:2, was derived fromthe following overlapping and/or extended nucleic acid sequences: IncyteClones 1614950 (BRAITUT12), 854869 (NGANNOT01), 1513925 (PANCTUT01), and1569880 (UTRSNOT05).

In one embodiment, the invention encompasses the novel humanubiquitin-conjugating enzyme, a polypeptide comprising the amino acidsequence of SEQ ID NO: 1, as shown in FIGS. 1A and 1B. UBCPB is 153amino acids in length and has a potential N-glycosylation site at aminoacid residue 24. UBCPB has chemical and structural homology with UbcH7(GI 1064916; SEQ ID NO:3) and UbcH5 (GI 460810; SEQ ID NO:4; FIG. 2). Inparticular, UBCPB and UbcH7 share 55% identity. UBCPB contains aconserved active site cysteine residue at amino acid 86 (FIG. 2). Asillustrated by FIGS. 3 and 4, UBCPB and UbcH7 have rather similarhydrophobicity plots.

The invention also encompasses UBCPB variants. A preferred UBCPB variantis one having at least 80%, and more preferably 90%, amino acid sequencesimilarity to the UBCPB amino acid sequence (SEQ ID NO: 1). A mostpreferred UBCPB variant is one having at least 95% amino acid sequencesimilarity to SEQ ID NO:1.

The invention also encompasses polynucleotides which encode UBCPB.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of UBCPB can be used to generate recombinant molecules whichexpress UBCPB. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A and 1B.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding UBCPB, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring UBCPB, and all such variations are to beconsidered as being specifically disclosed.

Although nucleotide sequences which encode UBCPB and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring UBCPB under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding UBCPB or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding UBCPB and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode UBCPB and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding UBCPB or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

Altered nucleic acid sequences encoding UBCPB which are encompassed bythe invention include deletions, insertions, or substitutions ofdifferent nucleotides resulting in a polynucleotide that encodes thesame or a functionally equivalent UBCPB. The encoded protein may alsocontain deletions, insertions, or substitutions of amino acid residueswhich produce a silent change and result in a functionally equivalentUBCPB. Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of UBCPB is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe genes encoding UBCPB. As used herein, an "allele" or "allelicsequence" is an alternative form of the gene which may result from atleast one mutation in the nucleic acid sequence. Alleles may result inaltered mRNAs or polypeptides whose structure or function may or may notbe altered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding UBCPB may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDER™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER™ and SEQUENCE NAVIGATOR™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode UBCPB, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of UBCPB in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressUBCPB.

As will be understood by those of skill in the art, it may beadvantageous to produce UBCPB-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter UBCPBencoding sequences for a variety of reasons, including but not limitedto, alterations which modify the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRpreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding UBCPB may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of UBCPB activity, it may be useful toencode a chimeric UBCPB protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the UBCPB encoding sequence and theheterologous protein sequence, so that UBCPB may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding UBCPB may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of UBCPB, or a portion thereof.For example, peptide synthesis can be performed using varioussolid-phase techniques (Roberge, J. Y. et al. (1995) Science269:202-204) and automated synthesis may be achieved, for example, usingthe ABI 431 A Peptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of UBCPB, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active UBCPB, the nucleotidesequences encoding UBCPB or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding UBCPB andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding UBCPB. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT® phagemid (Stratagene,LaJolla, Calif.) or PSPORT™ plasmid (Gibco BRL) and the like may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding UBCPB,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for UBCPB. For example, when largequantities of UBCPB are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT® (Stratagene), in which the sequence encoding UBCPB may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of 3-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression ofsequences encoding UBCPB may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CAMVmay be used alone or in combination with the omega leader sequence fromTMV (Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R.et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) ResultsProbl. Cell Differ. 17:85-105). These constructs can be introduced intoplant cells by direct DNA transformation or pathogen-mediatedtransfection. Such techniques are described in a number of generallyavailable reviews (see, for example, Hobbs, S. or Murry, L. E. in McGrawHill Yearbook of Science and Technology (1992) McGraw Hill, New York,N.Y.; pp. 191-196.

An insect system may also be used to express UBCPB. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding UBCPB may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of UBCPB will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which UBCPB may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding UBCPB may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing UBCPB in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding UBCPB. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding UBCPB, its initiation codon, and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressUBCPB may be transformed using expression vectors which may containviral origins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 1 1:223-32) and adeninephosphoribosyltransferase (Lowy, 1. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).

Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-13 1).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding UBCPB isinserted within a marker gene sequence, recombinant cells containingsequences encoding UBCPB can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding UBCPB under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding UBCPB and express UBCPB may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein.

The presence of polynucleotide sequences encoding UBCPB can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding UBCPB. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding UBCPB to detect transformantscontaining DNA or RNA encoding UBCPB. As used herein "oligonucleotides"or "oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofUBCPB, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson UBCPB is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding UBCPB includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, the sequences encoding UBCPB, orany portions thereof may be cloned into a vector for the production ofan mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in Vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits (Pharmacia & Upjohn, (Kalamazoo, Mich.);Promega (Madison, Wis.); and U.S. Biochemical Corp., Cleveland, Ohio).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding UBCPB may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeUBCPB may be designed to contain signal sequences which direct secretionof UBCPB through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding UBCPBto nucleotide sequence encoding a polypeptide domain which willfacilitate purification of soluble proteins. Such purificationfacilitating domains include, but are not limited to, metal chelatingpeptides such as histidine-tryptophan modules that allow purification onimmobilized metals, protein A domains that allow purification onimmobilized immunoglobulin, and the domain utilized in the FLAGSextension/affinity purification system (Immunex Corp., Seattle, Wash.).The inclusion of cleavable linker sequences such as those specific forFactor XA or enterokinase (Invitrogen, San Diego, Calif.) between thepurification domain and UBCPB may be used to facilitate purification.One such expression vector provides for expression of a fusion proteincontaining UBCPB and a nucleic acid encoding 6 histidine residuespreceding a thioredoxin or an enterokinase cleavage site. The histidineresidues facilitate purification on IMIAC (immobilized metal ionaffinity chromatography as described in Porath, J. et al. (1992, Prot.Exp. Purif. 3: 263-281) while the enterokinase cleavage site provides ameans for purifying UBCPB from the fusion protein. A discussion ofvectors which contain fusion proteins is provided in Kroll, D. J. et al.(1993; DNA Cell Biol. 12:441-453).

In addition to recombinant production, fragments of UBCPB may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431 APeptide Synthesizer (Perkin Elmer). Various fragments of UBCPB may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule

Therapeutics

In other embodiments of the invention, UBCPB or fragments thereof may beused for therapeutic purposes.

Based on the chemical and structural homology among UBCPB (SEQ ID NO:1), UbcH7 (SEQ ID NO:3), and UbcH5 (SEQ ID NO:4), UBCPB appears to havea role in disorders involving protein degradation, such as Alzheimer'sdisease, muscle wasting syndrome, viral infections, and cancer.

In one embodiment, UBCPB, fragments, or derivatives thereof may beadministered to a subject in order to effect the degradation of harmfulproteins in any disorder in which degradation of harmful proteins isdesired. In one aspect, UBCPB or an UBCPB derivative can be administeredto effect the degradation of the specific proteins that may eitherinitiate the onset or mediate the debilitating effects of Alzheimer'sdisease, such as amyloid beta-protein precursor. In addition, usingconventional methods, UBCPB may be altered in such a way as tospecifically target and therefore degrade potentially harmful proteins,such as APP, in Alzheimer's disease patients.

In another embodiment, UBCPB or derivatives thereof may be modified byrandom mutagenesis or other means and then assayed for specific bindingto a desired target protein.

The UBCPB or derivatives are thus engineered to target foreign proteinsfor degradation. Examples of target proteins include, but are notlimited to viral proteases, capsids, and integrases made by HIV, herpessimplex virus, adenovirus. The engineered UBCPB may be administered to asubject to treat viral infections including but not limited to herpessimplex virus, Epstein-Barr virus, adenovirus, rhinovirus, and HIV.

In another embodiment, antagonists or inhibitors of the protein, or avector expressing the antisense of the polynucleotide sequence encodingUBCPB may be administered to a subject in order to inhibit or modulateUBCPB-associated protein degradation, thereby treating disorders orconditions where inhibition or modulation is desired. For example, suchantagonists or inhibitors may be used to stop UBCPB-directed degradationof p53 protein, thus allowing p53 to suppress tumor development.

In another embodiment, antagonists or inhibitors of the protein, or avector expressing the antisense of the polynucleotide sequences encodingUBCPB may be administered to a subject in order to prevent thewidespread degradation of proteins that occurs in such disorders asmuscle wasting syndrome.

In another embodiment, other inhibitors which block or modulate theeffects of UBCPB, such as protein degradation, may be used in thosesituations where such inhibition is therapeutically desirable. Suchinhibitors may be produced using methods which are generally known inthe art. A particular method involves the use of purified UBCPB toproduce antibodies or to screen libraries of pharmaceutical agents forthose which specifically bind UBCPB. In one aspect, antibodies which arespecific for UBCPB may be used directly as an antagonist, or indirectlyas a targeting or delivery mechanism for bringing a pharmaceutical agentto cells or tissue which express UBCPB.

The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith UBCPB or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to UBCPB have an amino acid sequence consisting of atleast five amino acids, and more is preferably at least 10 amino acids.It is also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of UBCPB amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

Monoclonal antibodies to UBCPB may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495497; Kozbor, D. et al.(1985) J. Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1984) Mol. Cell Biol.62:109-120).

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceUBCPB-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobin libraries (BurtonD. R. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

Antibody fragments which contain specific binding sites for UBCPB mayalso be generated. For example, such fragments include, but are notlimited to, the F(ab')2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab')2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between UBCPB and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering UBCPB epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingUBCPB, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding UBCPB may be used in situations in which it would be desirableto block the transcription of the mRNA. In particular, cells may betransformed with sequences complementary to polynucleotides encodingUBCPB. Thus, antisense molecules may be used to modulate UBCPB activity,or to achieve regulation of gene function. Such technology is now wellknown in the art, and sense or antisense oligomers or larger fragments,can be designed from various locations along the coding or controlregions of sequences encoding UBCPB.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingUBCPB. These techniques are described both in Sambrook et al. (supra)and in Ausubel et al. (supra).

Genes encoding UBCPB can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide orfragment thereof which encodes UBCPB. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector and even longer if appropriate replicationelements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding UBCPB, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using "triple helix" base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding UBCPB.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding UBCPB. Such DNA sequences may be incorporatedinto a wide variety of vectors with suitable RNA polymerase promoterssuch as T7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysuitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of UBCPB, antibodies toUBCPB, mimetics, agonists, antagonists, or inhibitors of UBCPB. Thecompositions may be administered alone or in combination with at leastone other agent, such as stabilizing compound, which may be administeredin any sterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans. In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1 %-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of UBCPB, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example UBCPB or fragments thereof, antibodies of UBCPB,agonists, antagonists or inhibitors of UBCPB, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind UBCPB may beused for the diagnosis of conditions or diseases characterized byexpression of UBCPB, or in assays to monitor patients being treated withUBCPB, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for UBCPB includemethods which utilize the antibody and a label to detect UBCPB in humanbod y fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuringUBCPB are known in the art and provide a basis for diagnosing altered orabnormal levels of UBCPB expression. Normal or standard values for UBCPBexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toUBCPB under conditions suitable for complex formation The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric, means. Quantities of UBCPB expressed insubject samples, control and disease from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingUBCPB may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, antisense RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofUBCPB may be correlated with disease. The diagnostic assay may be usedto distinguish between absence, presence, and excess expression ofUBCPB, and to monitor regulation of UBCPB levels during therapeuticintervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding UBCPB or closely related molecules, may be used to identifynucleic acid sequences which encode UBCPB. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding UBCPB, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe UBCPB encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring UBCPB.

Means for producing specific hybridization probes for DNAs encodingUBCPB include the cloning of nucleic acid sequences encoding UBCPB orUBCPB derivatives into vectors for the production of mRNA probes. Suchvectors are known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding UBCPB may be used for the diagnosis ofconditions or diseases which are associated with expression of UBCPB.Examples of such conditions or diseases include cancers of the stomach,brain, large intestine, parathyroid, ureter, pancreas, breast, andparaganglion. The polynucleotide sequences encoding UBCPB may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pIN, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered UBCPB expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequences encoding UBCPB may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingUBCPB may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding UBCPB in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of UBCPB, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes UBCPB, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding UBCPB may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5'→3') and another with antisense(3←5'), employed under optimized conditions for identification of aspecific gene or condition. The same two oligomers, nested sets ofoligomers, or even a degenerate pool of oligomers may be employed underless stringent conditions for detection and/or quantitation of closelyrelated DNA or RNA sequences.

Methods which may also be used to quantitate the expression of UBCPBinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or colorimetricresponse gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequences whichencode UBCPB may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequences may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma et al. (1988) Human Chromosomes: A ManualTechniques, Pergamon Press, New York, N.Y.) may be correlated with otherphysical chromosome mapping techniques and genetic map data. Examples ofgenetic map data can be found in the 1994 Genome Issue of Science(265:1981 f). Correlation between the location of the gene encodingUBCPB on a physical chromosomal map and a specific disease, orpredisposition to a specific disease, may help delimit the region of DNAassociated with that genetic disease. The nucleotide sequences of thesubject invention may be used to detect differences in gene sequencesbetween normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, UBCPB, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenUBCPB and the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT application WO84/03564. In this method, as applied to UBCPB large numbers of differentsmall test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The test compounds are reacted withUBCPB, or fragments thereof, and washed. Bound UBCPB is then detected bymethods well known in the art. Purified UBCPB can also be coateddirectly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding UBCPB specificallycompete with a test compound for binding UBCPB. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with UBCPB.

In additional embodiments, the nucleotide sequences which encode UBCPBmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I BRAITUT12 cDNA Library Construction

The BRAITUT12 cDNA library was constructed from cancerous brain tissue.Both tissues were obtained from a 40-year-old Caucasian female duringcerebral meningeal excision following diagnosis of grade 4 (of 4)gemistocytic astrocytoma localized in the left frontal part of thebrain. Prior to surgery the patient was also diagnosed with coma,epilepsy, and paralysis. Prior to surgery the patient was prescribedDECADRON® (dexamethasone; Merck & Co., Inc. West Point, Pa.), andphenytoin sodium. Patient's history included a diagnosis of chronicnephritis.

The frozen tissue was homogenized and lysed using a BrinkmannHomogenizer Polytron PT-3000 (Brinkmann Instruments, Westbury, N.J.) inguanidinium isothiocyanate solution. The lysate was centrifuged over a5.7 M CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments) for 18 hours at 25,000 rpm atambient temperature. The RNA was extracted with acid phenol pH 4.7,precipitated using 0.3 M sodium acetate and 2.5 volumes of ethanol,resuspended in RNAse-free water, and DNase treated at 37° C. The RNAextraction was repeated with acid phenol pH 4.7 and precipitated withsodium acetate and ethanol as before. The mRNA was then isolated usingthe Qiagen Oligotex kit (QIAGEN, Inc.; Chatsworth, Calif.) and used toconstruct the cDNA library.

The mRNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning (Cat.#18248-013; Gibco/BRL).

The commercial plasmid PSPORT 1 (Gibco/BRL) was digested with EcoRIrestriction enzyme (New England Biolabs, Beverley, Mass.). Theoverhanging ends of the plasmid were filled in using Klenow enzyme (NewEngland Biolabs) and 2'-deoxynucleotide 5'-triphosphates (dNTPs). Theplasmid was self-ligated and transformed into the bacterial host, E.coli strain JM 109. An intermediate plasmid produced by the bacteriafailed to digest with EcoRI confirming the desired loss of the EcoRIrestriction site.

This intermediate plasmid (PSPORT 1-ΔRI) was then digested with Hind IIIrestriction enzyme (New England Biolabs) and the overhang was filled inwith Klenow and dNTPs. A 1O-mer linker of sequence 5' . . . CGGAATTCCG .. . 3' was phosphorylated and ligated onto the blunt ends. The productof the ligation reaction was digested with EcoR I and self-ligated.Following transformation into JM 109 host cells, plasmids were isolatedand screened for the digestibility with EcoR I but not with Hind III. Asingle colony which met this criteria was designated pINCY 1. Theplasmid produced by this colony was sequenced and found to containseveral copies of the 1O-mer linker. These extra linkers did not presenta problem as they were eliminated when the vector was prepared forcloning.

The plasmid was tested for its ability to incorporate cDNAs from alibrary prepared using Not I and EcoR I restriction enzymes. Severalclones were sequenced and a single clone containing an insert ofapproximately 0.8 kb was selected to prepare a large quantity of theplasmid for library production. After digestion with Not I and EcoR I,the plasmid and the cDNA insert were isolated on an agarose gel and thevector was purified on a QIAQUICK™ (QIAGEN) column for use in libraryconstruction.

cDNAs were fractionated on a Sepharose CL4B column (Cat. #275105-01;Pharmacia), and those cDNAs exceeding 400 bp were ligated into PSPORT I.The plasmid PSPORT I was subsequently transformed into DH5A™ competentcells (Cat. #18258-012; Gibco/BRL).

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the REAL Prep96 Plasmid Kit for Rapid Extraction Alkaline Lysis Plasmid Minipreps(Catalog #26173; QIAGEN). This kit enabled the simultaneous purificationof 96 samples in a 96-well block using multi-channel reagent dispensers.The recommended protocol was employed except for the followingchanges: 1) the bacteria were cultured in 1 ml of sterile Terrific Broth(Catalog #22711, LIFE TECHNOLOGIES™) with carbenicillin at 25 mg/L andglycerol at 0.4%; 2) after inoculation, the cultures were incubated for19 hours and at the end of incubation, the cells were lysed with 0.3 mlof lysis buffer; and 3) following isopropanol precipitation, the plasmidDNA pellet was resuspended in 0.1 ml of distilled water. After the laststep in the protocol, samples were transferred to a 96-well block forstorage at 4° C.

The cDNAs were sequenced by the method of Sanger et al. (1975, J. Mol.Biol. 94:441 f), using a Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.)in combination with Peltier Thermal Cyclers (PTC200 from MJ Research,Watertown, Mass.) and Applied Biosystems 377 DNA Sequencing Systems.

Most of the sequences disclosed herein were sequenced according tostandard ABI protocols, using ABI kits (Cat. Nos. 79345, 79339, 79340,79357, 79355). The solution volumes were used at 0.25×1.0×concentrations. Some of the sequences disclosed herein were sequencedusing different solutions and dyes which, unless otherwise noted, camefrom Amersham Life Science (Cleveland, Ohio).

First, stock solutions were prepared with HPLC water. The followingsolutions were each mixed by vortexing for 2 min: I) TRIS-EDTA (TE)Buffer was prepared by adding 49 ml water to 1 ml 50× TRIS-EDTAconcentrate, and 2) 10% Reaction Buffer was prepared by adding 45 mlwater to 5 ml Concentrated Thermo Sequenase (TS) Reaction Buffer.

Second, 0.2 μM energy transfer (ET) primers were prepared in thefollowing manner. Each primer tube was centrifuged prior to opening toassure that all primer powder was on the bottom of the tube. After eachsolubilization step, the mixture was vortexed for 2 min and thencentrifuged for about 10 sec in a table-top centrifuge. 1 ml of 1× TEwas added to each primer powder; adenine and cytosine dissolved primers(5-carboxyrhodamine-6G (R6G) and 6-carboxyfluorescein (FAM),respectively), were diluted with 9 ml lx TE. Guanine and thymine dyes(N,N,N',N"-tetramethyl-6-carboxyrhodamine (TAM) and6-carboxy-X-rhodamine (ROX), respectively) were diluted with 19 ml 1×TE.

Next, the sequencing reaction ready mix was prepared as follows: 1)nucleotides A and C (8 ml of each) were added to 6 ml ET primer and 18ml TS reaction buffer; and 2) nucleotides G and T (8 ml of each) wereadded to 6 ml ET primer and 18 ml TS reaction buffer.

After vortexing for 2 min and centrifuging for 20 sec, the resultingsolution was divided into tubes in volumes of 8 ml per tube in order tomake 1× (A,C) and 2× (G,T) solutions.

Prior to thermal cycling, each nucleotide was individually mixed withDNA template in the following proportions:

    ______________________________________                                        Reagent       A(μL)                                                                              C(μL) G(μL)                                                                            T(μL)                                 ______________________________________                                        Reaction ready premix                                                                       2       2        4     4                                        DNA template  1       1        2     2                                        Total Volume  3       3        6     6                                        ______________________________________                                    

These solutions undergo the usual thermal cycling:

1. Rapid thermal ramp to 94° C. (94° C. for 20 sec)*

2. Rapid thermal ramp to 50° C. (50° C. for 40 sec)*

3. Rapid thermal ramp to 68° C. (68° C. for 60 sec)*

* Steps 1, 2, and 3 were repeated for 15 cycles

4. Rapid thermal ramp to 94° C. (94° C. for 20 sec)**

5. Rapid thermal ramp to 68° C. (68° C. for 60 sec)**

** Steps 4 and 5 were repeated for 15 cycles

6. Rapid thermal ramp to 4° C. and hold until ready to combine.

After thermal cycling, the A, C, G, and T reactions with each DNAtemplate were combined. Then, 50 μL 100% ethanol was added and thesolution was spun at 4° C. for 30 min. The supematant was decanted andthe pellet was rinsed with 100 μL 70% ethanol. After being spun for 15min the supernatant was discarded and the pellet was dried for 15 minunder vacuum. The DNA sample was dissolved in 3 μL of formamide/50 mMEDTA. The resulting samples were loaded on wells in volumes of 2 μL perwell for sequencing in ABI sequencers.

III Homology Searching of cDNA Clones and Their Deduced Proteins

After the reading frame was determined, the nucleotide sequences of theSequence Listing or amino acid sequences deduced from them were used asquery sequences against databases such as GenBank, SwissProt, BLOCKS,and Pima II. These databases which contain previously identified andannotated sequences, were searched for regions of homology (similarity)using BLAST, which stands for Basic Local Alignment Search Tool(Altschul S. F. (1993) J. Mol. Evol. 36:290-300; Altschul, S. F. et al.(1990) J. Mol. Biol. 215:403-10).

BLAST produced alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST was especially useful in determining exact matches orin identifying homologs which may be of prokaryotic (bacterial) oreukaryotic (animal, fungal, or plant) origin. Other algorithms such asthe one described in Smith et al. (1992, Protein Engineering 5:35-51),incorporated herein by reference, could have been used when dealing withprimary sequence patterns and secondary structure gap penalties. Thesequences disclosed in this application have lengths of at least 49nucleotides, and no more than 12% uncalled bases (where N is recordedrather than A, C, G,or T).

The BLAST approach, as detailed in Karlin et al. (supra) andincorporated herein by reference, searched for matches between a querysequence and a database sequence. BLAST evaluated the statisticalsignificance of any matches found, and reported only those matches thatsatisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides.

Incyte nucleotide sequences were searched against the GenBank databasesfor primate (pri), rodent (rod), and other mammalian sequences (mam);and deduced amino acid sequences from the same clones were then searchedagainst GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp), and eukaryote (eukp) for homology. The relevantdatabase for a particular match were reported as GIxxx±p (where xxx ispri, rod, etc. and if present, p=peptide). The product score wascalculated as follows: the % nucleotide or amino acid identity [betweenthe query and reference sequences] in BLAST is multiplied by the %maximum possible BLAST score [based on the lengths of query andreference sequences] and then divided by 100. Where an Incyte Clone washomologous to several sequences, up to five matches were provided withtheir relevant scores. In an analogy to the hybridization proceduresused in the laboratory, the electronic stringency for an exact match wasset at 70, and the conservative lower limit for an exact match was setat approximately 40 (with 1-2% error due to uncalled bases).

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S.F. 1993 and 1990,supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding UBCPB occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of UBCPB-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

Full length UBCPB-encoding nucleic acid sequence (SEQ ID NO:2) is usedto design oligonucleotide primers for extending a partial nucleotidesequence to full length or for obtaining 5' or 3', intron or othercontrol sequences from genomic libraries. One primer is synthesized toinitiate extension in the antisense direction (XLR) and the other issynthesized to extend sequence in the sense direction (XLF). Primers areused to facilitate the extension of the known sequence "outward"generating amplicons containing new, unknown nucleotide sequence for theregion of interest. The initial primers are designed from the cDNA usingOLIGO 4.06 (National Biosciences), or another appropriate program, to be22-30 nucleotides in length, to have a GC content of 50% or more, and toanneal to the target sequence at temperatures about 68°-72° C. Anystretch of nucleotides which would result in hairpin structures andprimer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters:

    ______________________________________                                        Step 1       94° C. for 1 min (initial denaturation)                   Step 2       65° C. for 1 min                                          Step 3       68° C. for 6 min                                          Step 4       94° C. for 15 sec                                         Step 5       65° C. for 1 min                                          Step 6       68° C. for 7 min                                          Step 7       Repeat step 4-6 for 15 additional cycles                         Step 8       94° C. for 15 sec                                         Step 9       65° C. for 1 min                                          Step 10      68° C. for 7:15 min                                       Step 11      Repeat step 8-10 for 12 cycles                                   Step 12      72° C. for 8 min                                          Step 13      4° C. (and holding)                                       ______________________________________                                    

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQUICK™ (QIAGEN). After recovery of the DNA, Kienow enzyme isused to trim single-stranded, nucleotide overhangs creating blunt endswhich facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E.coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

    ______________________________________                                        Step 1      94° C. for 60 sec                                          Step 2      94° C. for 20 sec                                          Step 3      55° C. for 30 sec                                          Step 4      72° C. for 90 sec                                          Step 5      Repeat steps 2-4 for an additional 29 cycles                      Step 6      72° C. for 180 sec                                         Step 7      4° C. (and holding)                                        ______________________________________                                    

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³² P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N. H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules to the UBCPB-encoding sequence, or any part thereof,is used to inhibit in vivo or in vitro expression of naturally occurringUBCPB. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on the codingsequences of UBCPB, as shown in FIGS. 1A and 1B, is used to inhibitexpression of naturally occurring UBCPB. The complementaryoligonucleotide is designed from the most unique 5' sequence as shown inFIGS. 1A and 1B and used either to inhibit transcription by preventingpromoter binding to the upstream nontranslated sequence or translationof an UBCPB-encoding transcript by preventing the ribosome from binding.Using an appropriate portion of the signal and 5' sequence of SEQ IDNO:2, an effective antisense oligonucleotide includes any 15-20nucleotides spanning the region which translates into the signal or 5'coding sequence of the polypeptide as shown in FIGS. 1A and 1B.

VIII Expression of UBCPB

Expression of UBCPB is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, PSPORT, previously used for thegeneration of the cDNA library, is used to express UBCPB in E. coli.Upstream of the cloning site, this vector contains a promoter forB-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion ofUBCPB into the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of UBCPB Activity

UBCPB activity can be assessed by measuring the ability to formthioester adducts with ubiquitin in the presence of E1 (Jentsch S.(1992) Ann. Rev. Genet. 26: 177-205). Nuber et al. (supra) describe onesuch procedure. Reaction mixtures include approximately 5-10 ng of E1, 1μg of ³² P-labeled GST-ubiquitin, 100 ng of UBCPB, a negative, or apositive E1 control in 30 mM TRIS-HCl, pH 7.6, 50 mM NaCl, 4 mM ATP, 10mM MgCl₂, 0.2 mM dithiothreitol. After 5 min at 25° C., reactions areterminated by incubating the mixtures for 15 min at 30° C. in 50 mMTRIS-HCl, pH 6.8, 4 M urea, 2% SDS, 10% glycerol. The whole reactionmixtures are separated on 10% SDS-polyacrylamide gels at 4° C. andradioactively labeled bands visualized by autoradiography.

X Production of UBCPB Specific Antibodies

UBCPB that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al (supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431 A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring UBCPB Using Specific Antibodies

Naturally occurring or recombinant UBCPB is substantially purified byimmunoaffinity chromatography using antibodies specific for UBCPB. Animmunoaffinity column is constructed by covalently coupling UBCPBantibody to an activated chromatographic resin, such as CnBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing UBCPB is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof UBCPB (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/UBCPB binding (eg, a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and UBCPB iscollected.

XII Identification of Molecules Which Interact with UBCPB

UBCPB or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled UBCPB, washed and any wells withlabeled UBCPB complex are assayed. Data obtained using differentconcentrations of UBCPB are used to calculate values for the number,affinity, and association of UBCPB with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 4                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 153 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: Consensus                                                        (B) CLONE: Consensus                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Met Met Xaa Ser Met Arg Val Val Lys Glu Le - #u Glu Asp Leu Gln Lys         #                15                                                           - Lys Pro Pro Pro Tyr Leu Arg Asn Leu Ser Se - #r Asp Asp Ala Asn Val         #            30                                                               - Leu Val Trp His Ala Leu Leu Leu Pro Asp Gl - #n Pro Pro Tyr His Leu         #        45                                                                   - Lys Ala Phe Asn Leu Arg Ile Ser Phe Pro Pr - #o Glu Tyr Pro Phe Lys         #    60                                                                       - Pro Pro Met Ile Lys Phe Thr Thr Lys Ile Ty - #r His Pro Asn Val Asp         #80                                                                           - Glu Asn Gly Gln Ile Cys Leu Pro Ile Ile Se - #r Ser Glu Asn Trp Lys         #                95                                                           - Pro Cys Thr Lys Thr Cys Gln Val Leu Glu Al - #a Leu Asn Val Leu Val         #           110                                                               - Asn Arg Pro Asn Ile Arg Glu Pro Leu Arg Me - #t Asp Leu Ala Asp Leu         #       125                                                                   - Leu Thr Gln Asn Pro Glu Leu Phe Arg Lys As - #n Ala Glu Glu Phe Thr         #   140                                                                       - Leu Arg Phe Gly Val Asp Arg Pro Ser                                         145                 1 - #50                                                   - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 547 base                                                          (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: Consensus                                                        (B) CLONE: Consensus                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - CGGCTCGAGG CCACGGGTGC CACACACTCG GTCCCGACAT GATGGNGAGC AT - #GCGAGTGG         60                                                                          - TGAAGGAGCT GGAGGATCTT CAGAAGAAGC CTCCCCCATA CCTGCGGAAC CT - #GTCCAGCG        120                                                                          - ATGATGCCAA TGTCCTGGTG TGGCACGCTC TCCTCCTACC CGACCAACCT CC - #CTACCACC        180                                                                          - TGAAAGCCTT CAACCTGCGC ATCAGCTTCC CGCCGGAGTA TCCGTTCAAG CC - #TCCCATGA        240                                                                          - TCAAATTCAC AACCAAGATC TACCACCCCA ACGTGGACGA GAACGGACAG AT - #TTGCCTGC        300                                                                          - CCATCATCAG CAGTGAGAAC TGGAAGCCTT GCACCAAGAC TTGCCAAGTC CT - #GGAGGCCC        360                                                                          - TCAATGTGCT GGTGAATAGA CCGAATATCA GGGAGCCCCT GCGGATGGAC CT - #CGCTGACC        420                                                                          - TGCTGACACA GAATCCGGAG CTGTTCAGAA AGAATGCCGA AGAGTTCACC CT - #CCGATTCG        480                                                                          - GAGTGGACCG GCCCTCCTAA CTCATGTTCT GACCCTCTGT GCACTGGATC CT - #CGGCATAG        540                                                                          #         547                                                                 - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 154 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: GenBank                                                          (B) CLONE: 1064916                                                  -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Met Ala Ala Ser Arg Arg Leu Met Lys Glu Le - #u Glu Glu Ile Arg Lys         #                15                                                           - Cys Gly Met Lys Asn Phe Arg Asn Ile Gln Va - #l Asp Glu Ala Asn Leu         #            30                                                               - Leu Thr Trp Gln Gly Leu Ile Val Pro Asp As - #n Pro Pro Tyr Asp Lys         #        45                                                                   - Gly Ala Phe Arg Ile Glu Ile Asn Phe Pro Al - #a Glu Tyr Pro Phe Lys         #    60                                                                       - Pro Pro Lys Ile Thr Phe Lys Thr Lys Ile Ty - #r His Pro Asn Ile Asp         #80                                                                           - Glu Lys Gly Gln Val Cys Leu Pro Val Ile Se - #r Ala Glu Asn Trp Lys         #                95                                                           - Pro Ala Thr Lys Thr Asp Gln Val Ile Gln Se - #r Leu Ile Ala Leu Val         #           110                                                               - Asn Asp Pro Gln Pro Glu His Pro Leu Arg Al - #a Asp Leu Ala Glu Glu         #       125                                                                   - Tyr Ser Lys Asp Arg Lys Lys Phe Cys Lys As - #n Ala Glu Glu Phe Thr         #   140                                                                       - Lys Lys Tyr Gly Glu Lys Arg Pro Val Asp                                     145                 1 - #50                                                   - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 147 amino                                                         (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -    (vii) IMMEDIATE SOURCE:                                                            (A) LIBRARY: GenBank                                                          (B) CLONE: 460810                                                   -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Met Ala Leu Lys Arg Ile Gln Lys Glu Leu Se - #r Asp Leu Gln Arg Asp         #                15                                                           - Pro Pro Ala His Cys Ser Ala Gly Pro Val Gl - #y Asp Asp Leu Phe His         #            30                                                               - Trp Gln Ala Thr Ile Met Gly Pro Pro Asp Se - #r Ala Tyr Gln Gly Gly         #        45                                                                   - Val Phe Phe Leu Thr Val His Phe Pro Thr As - #p Tyr Pro Phe Lys Pro         #    60                                                                       - Pro Lys Ile Ala Phe Thr Thr Lys Ile Tyr Hi - #s Pro Asn Ile Asn Ser         #80                                                                           - Asn Gly Ser Ile Cys Leu Asp Ile Leu Arg Se - #r Gln Trp Ser Pro Ala         #                95                                                           - Leu Thr Val Ser Lys Val Leu Leu Ser Ile Cy - #s Ser Leu Leu Cys Asp         #           110                                                               - Pro Asn Pro Asp Asp Pro Leu Val Pro Asp Il - #e Ala Gln Ile Tyr Lys         #       125                                                                   - Ser Asp Lys Glu Lys Tyr Asn Arg His Ala Ar - #g Glu Trp Thr Gln Lys         #   140                                                                       - Tyr Ala Met                                                                 145                                                                           __________________________________________________________________________

What is claimed is:
 1. A substantially purified ubiquitin-conjugatingenzyme comprising the amino acid sequence of SEQ ID NO:1.
 2. Apharmaceutical composition comprising a substantially purifiedubiquitin-conjugating enzyme of claim 1 in conjunction with a suitablepharmaceutical carrier.